Sensor calibration

ABSTRACT

A process for calibrating a glucose sensor under sterile conditions includes providing separate, sterile, glucose-containing calibration fluids, each having a different glucose concentration, and in turn providing these fluids to a sensing zone containing a sensing probe of a glucose sensor. Each solution is typically, in turn, propelled into the sensing zone, thus flushing out used fluid already present in the sensing zone. The process provides rapid calibration of a glucose sensor in a sterile fashion and is therefore appropriate for point-of-use calibration.

PRIORITY CLAIM AND CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/592,266 filed Oct. 3, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/115,967, filed Aug. 2, 2016, now U.S. Pat. No.10,433,778, issued Oct. 8, 2019, which is a National Stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/GB2015/050283, filed Feb. 3, 2015, which claims the benefit ofpriority to GB 1401878.2, filed Feb. 4, 2014. The disclosure of theprior applications are considered part of (and are incorporated byreference in) the disclosure of this application.

FIELD

Described herein is a process for calibrating a glucose sensor understerile conditions in a manner which is appropriate for point of usecalibration; a calibration unit and glucose sensor kit including thecalibration unit as well as a glucose sensor probe; and a process forthe preparation of a sterile calibration unit.

BACKGROUND

The usual aim in developing a chemical sensor or biosensor is to producea signal, which is proportional to the concentration of a specificchemical or set of chemicals (analyte). The sensor usually has two maincomponents, a chemical or biological part that reacts or complexes withthe analyte in question (ideally specifically) to form new chemical orbiological products or changes in energy that can be detected by meansof the second component, a transducer. The chemical/biological componentcan be said to act as a receptor/indicator for the analyte. A variety oftransduction methods can be used including electrochemical (such aspotentiometric, amperometric, conductimetric, impedimetric), optical,calorimetric and acoustic. After transduction the signal is usuallyconverted to an electronic digital signal.

Since the signal generated by the chemical/biological reaction with theanalyte is usually dependent not only on the concentration of theanalyte but also on the characteristics of the sensor itself, suchsensors usually require calibration before they can be utilisedquantitatively. The way in which the signal varies with the analyteconcentration determines the shape of the calibration curve (signalversus analyte concentration) and may define the number of calibrationpoints. Typical calibration curves can be straight line, exponential,s-shaped etc. and the principal of calibration applies to allmethodologies of transduction for chemical or biological sensors.

Calibration of sensors with an invasive medical application has its ownset of specific issues. Invasive or implantable medical sensors are tobe presented to the patient in a sterile condition, and are often singleuse, disposable devices. Ideally, the sensor should be calibrated justbefore its use since some sensor characteristics that can affect thecalibration curve vary with time (ageing effect). It is often the casethat the time between sensor manufacture and use can be many months, socalibration at the point of manufacture can lead to inaccuracies in theend result. This means that the attendant clinician or nurse will berequired to perform the calibration whilst maintaining sterility of thesensor. Additional constraints applied by the clinician/nurse are thatthe calibration process should be simple to perform, ideally invisibleto the person performing the calibration, and be quickly completed(preferably in less than 10 minutes).

Calibration of many currently available medical sensors requires theclinician/nurse to carry out a number of specific steps which can leadto errors or inaccuracies in the measurement if the process is notfollowed correctly. There is therefore a need for a more simplecalibration process, useful in connection with invasive or implantablesensors, which fulfils the above discussed requirements.

Sterilisation of such devices can also present difficulties. Thesterilisation process is typically carried out at the point ofmanufacture to avoid difficulties with poor or incomplete sterilisationprocedures at a hospital or clinic, and to save time on behalf of theclinician or nurse. In the case of glucose, however, glucose-containingsolutions have been found to degrade on sterilisation with either heator gamma-radiation. Thus, the pre-sterilisation of such solutions atmanufacture must be avoided. Glucose can instead be provided in solidform, since in this state sterilisation can be carried out withoutcausing degradation. However, the user is then required to make up therequired glucose-containing calibration solutions at the point of use.This adds additional steps to the procedure and can be time-consuming.For example over 45 minutes may be required to ensure that glucose hasfully dissolved and equilibrated in the solution.

Further, there is a need to maintain sterility during the calibration, afactor which is difficult to achieve given the differing sterilisationprocesses which may be needed for different parts of the calibrationunit and sensor. Sterility will be lost if the process requires the userto break open sterile packages in order to complete calibration.

There is therefore particularly a need for a means of calibrating aglucose sensor which can be carried out under sterile conditions,without loss of sterility during the process, and which can be carriedout in a short time.

SUMMARY

Provided herein is a process for calibrating a glucose sensor in asterile environment, the glucose sensor comprising a sensing probe fordetecting glucose, the process comprising: (i) providing a calibrationunit comprising (a) at least a first and a second sterile calibrationfluid, each calibration fluid having a different concentration ofglucose, (b) a sensing zone, a sensing region of the probe of theglucose sensor being located within the sensing zone, and (c) a wastechamber for collecting used calibration fluid; (ii) providing the firstcalibration fluid to the sensing zone such that the fluid is in contactwith the sensing region, and determining the sensor output; (iii)flushing the first calibration fluid to the waste chamber; (iv)providing the second calibration fluid to the sensing zone such that thefluid is in contact with the sensing region, and determining the sensoroutput; (v) using the sensor output readings to calibrate the glucosesensor; wherein the calibration process is carried out under sterileconditions.

Typically, said process is a process for calibrating an invasive fibreoptic glucose sensor containing a fluorescent glucose indicator system,the sensor probe being adapted to detect glucose in vivo.

Also provided is a glucose sensor kit comprising a sensing probe fordetecting glucose and a calibration unit, the calibration unitcomprising:

-   -   a sensing zone having at least one inlet and at least one        outlet, a sensing region of the probe being located in the        sensing zone;    -   a first calibration chamber containing a first sterile        calibration fluid, the first calibration chamber being connected        to, or adapted for connection to, an inlet to the sensing zone;    -   a second calibration chamber containing a second sterile        calibration fluid having a glucose concentration different from        that of the first calibration fluid, the second calibration        chamber being connected to, or adapted for connection to, an        inlet to the sensing zone;    -   at least one fluid propulsion means, e.g. manual or automated        propulsion means, for propelling calibration fluid from the        calibration chambers to the sensing zone;    -   a waste chamber arranged to collect used calibration fluid which        is flushed out of the sensing zone;    -   the glucose sensor kit being arranged to carry out calibration        of the sensor under sterile conditions.

Typically, the sensing probe comprised in said kit is part of aninvasive fibre optic glucose sensor containing a fluorescent glucoseindicator system, and the sensor probe is adapted to detect glucose invivo.

The process and kit described herein therefore provide for sterilecalibration, which can be completed in a short timescale and whilstmaintaining sterility. This is particularly beneficial where calibrationis carried out at point-of-use. In particular, where previous sterilecalibration processes relied upon calibration solutions being preparedby the user, the calibration methods, systems, and devices providedherein can avoid this time-consuming step by providing the glucosesolutions in ready to use form. This means that the user can directlyprovide the solutions to the sensing zone of the calibration unit, and areading can immediately be taken. Significant time savings in thecalibration carried out by the user can be made.

The ready-to-use sterile glucose calibration solutions can be preparedby a process comprising the steps:

(i) providing glucose in solid form in a first mixing chamber, and wateror an aqueous solution in a second mixing chamber;

(ii) sterilising the first and second mixing chambers by use of heat orirradiation;

(iii) mixing the contents of the first and second chambers to provide afirst, sterile glucose-containing calibration fluid;

(iv) providing at least a portion of the first calibration fluid to afirst calibration chamber, whilst maintaining sterility of the fluid;

(v) providing at least one further sterile calibration fluid, eachfurther fluid having a glucose concentration different from one anotherand from the first calibration fluid, the further calibration fluid(s)being provided in one or more further calibration chambers;

(vi) connecting the first and one or more further calibration chambersto a calibration unit such that the calibration chambers are connectedto, or adapted for connection to, an inlet to a sensing zone of thecalibration unit;

(vii) sterilising the calibration unit using a surface sterilant, saidsterilising step being carried out either before or after step (vi), toprovide a sterile calibration unit containing at least two sterilecalibration fluids.

The above process therefore provides a convenient manufacturing methodto provide ready-to-use sterile glucose calibration fluids within asterile calibration unit. The method described herein requires onlysimple equipment in order to prepare the sterile glucose containingsolutions. This has the advantage that equipment able to withstandsterilisation by heat (e.g. standard autoclaving) can be used, thusproviding a simple manufacturing process.

Also provided is a calibration unit for calibrating a glucose sensorcomprising:

-   -   a sensing zone having at least one inlet and at least one        outlet, the sensing zone being arranged to house a sensing        region of a sensing probe of the glucose sensor;    -   a first calibration chamber containing a first sterile        calibration fluid, the first calibration chamber being connected        to, or adapted for connection to, an inlet to the sensing zone;    -   a second calibration chamber containing a second sterile        calibration fluid having a glucose concentration different from        that of the first calibration fluid, the second calibration        chamber being connected to, or adapted for connection to, an        inlet to the sensing zone;    -   at least one fluid propulsion means, e.g. manual or automated        propulsion means, for propelling calibration fluid from the        calibration chambers to the sensing zone;    -   a waste chamber arranged to collect used calibration fluid which        is flushed out of the sensing zone;    -   the calibration unit being arranged to carry out calibration of        the sensor under sterile conditions.

Typically said calibration unit is suitable for calibrating an invasivefibre optic glucose sensor, said sensor containing a fluorescent glucoseindicator system and a sensor probe being adapted to detect glucose invivo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically depicts a glucose sensor kit as provided herein.

FIGS. 2 and 2 a schematically depict a sensor for calibration by theprocess described herein.

FIG. 3 provides a flow chart depicting the process for preparing asterile calibration unit as described herein.

FIG. 4 schematically depicts the process for preparing a sterilecalibration unit as described herein.

DETAILED DESCRIPTION

A glucose kit is described further with reference to the accompanyingdrawings which serve as an example and are not intended to limit thescope of the invention.

Reference is made herein to sterile calibration fluids and tocalibration being carried out under sterile conditions. The term sterileindicates that the fluid or conditions are free from living germs ormicroorganisms. Sterility as referred to herein can be achieved bystandard techniques of sterilisation well-known in the art, for exampleautoclave, gamma-radiation or chemical sterilisation, e.g. by ethyleneoxide.

Glucose Sensor

The calibration process described herein can be used with an invasive(including implantable) glucose sensor, for example a sensor whichdetermines glucose concentration in blood (e.g. intravascular), or ininterstitial tissue (e.g. subcutaneous). In some cases, the sensor probeis adapted to detect glucose in vivo. Such sensors are required to besterile immediately prior to use and accordingly if calibrated at pointof use, this should be done without loss of sterility. In some cases,the calibration process is for use with a fibre optic sensor including afluorescent indicator system. Such sensors and indicator systems areknown, for example, from U.S. Pat. No. 6,387,672 and WO 2012/095628. Thedescribed calibration process and kit can, however, be used with anytype of glucose sensor requiring sterile calibration.

One particular invasive glucose sensor is based on a fibre optictechnique and is depicted in FIG. 2 . The sensor 1 includes a probe 2which is adapted for insertion into a patient, for example insertioninto a blood vessel through a cannular. The probe includes a sensingregion 3, designed to contact a sample to be tested, (depicted in moredetail in FIG. 2 a ) in which the glucose indicator system 4, andtypically also a temperature sensor 5, are positioned. The glucoseindicating system is immobilised on or in an optical fibre 6, such thata signal emitted by the indicating system is transmitted through theoptical fibre. The optical fibre extends through cable 7 to connector 8,which is adapted to mate with a controller (e.g. as depicted in FIG. 1). The controller is typically connected to further optical cable thatmates with the connector at one end and at the other bifurcates toconnect to (a) an appropriate light source for the optical sensor and(b) a detector for the emitted signal. Electrical connection to thetemperature sensor is also provided through connector 8 and appropriatedetection equipment is provided by the monitor.

The probe may be provided as a disposable unit, adapted for connectionto a non-disposable controller. Thus, for example, the disposable unitmay include the probe 2, cable 7 and connector 8. The sensor kitdescribed herein can include a disposable probe unit including theprobe, cable and connector, the connector being arranged to opticallyconnect the probe unit to a controller.

The sensing region of the sensor can be surrounded by (or housed in) amembrane 9, which can be haemocompatible, or provided with ahaemocompatible coating, and can allow diffusion of glucose from thesurrounding blood or body fluid to the indicating system 4.

A suitable indicator system can include a boronic acid compound, e.g. adiboronic acid, acting as receptor and a fluorophore. A boronic acidspecies can provide the ability to complex with glucose and thefluorescence emission pattern of the fluorophore can be altered in thepresence of glucose, which can allow optical detection.

In some cases, the indicator system can be immobilised to the opticalfibre in a hydrogel which allows diffusion of water and glucose to thereceptor compound. Cross-linked polyacrylamide orpolyhydroxyethylmethacrylate (p-HEMA) are examples of hydrogels that canbe used.

Calibration Unit

A calibration unit according to one embodiment is depicted in FIG. 1 .The calibration unit can include a sensing zone SZ which is arranged tohouse the sensing region 3 of the probe P of glucose sensor GS. Thesensor is depicted in position in the sensing zone in FIG. 1 , but thecalibration unit may be provided without the probe within the housing.In this case, the probe is separately inserted, typically under sterileconditions. This may be done, for example, by inserting probe 3 throughseal 10. Further, FIG. 1 depicts probe 3 connected to cable 7 andultimately to controller CON. Alternatively, a disposable probe unitincluding probe 3, cable 7 and connector 8 (not depicted) may beprovided, the connector being adapted for later connection to a suitablecontroller.

The sensing zone SZ can be capable of containing fluid for glucosedetection. Thus, the sensing zone can be arranged to contain acalibration fluid whilst a sensor reading is taken using the sensingregion 3 of probe P. At least one inlet (IN) and at least one outlet(OUT) can be provided to enable calibration fluids to enter and exit thesensing zone.

In some embodiments the sensor is an invasive glucose sensor. In suchembodiments the probe P is configured to detect analyte in a samplewhile located within the body of a subject (e.g. not merely mounted ontissue such as the subject's skin). However, the calibration unit istypically not used to calibrate the sensor while the sensor is in vivo,because it is typically not desirable to introduce calibration fluidinto the body of a subject.

In order to be suitable for calibrating an invasive fibre optic glucosesensor, the calibration unit is typically configured to be removed fromthe invasive sensor prior to use of the sensor to detect analyte in asample. Thus, the presence of the calibration unit is typically notrequired in order for the invasive sensor to perform its function invivo. Typically, the sensing zone SZ of the calibrator is not the zonein which the sensing region 3 of probe P is located when performing itsfunction in vivo.

The calibration unit can include at least two calibration chambers. Asdepicted in FIG. 1 , three calibration chambers are preferred forcalibration of glucose. Glucose has a non-linear calibration curve, soaccurate calibration can be carried out by use of at least threecalibration fluids, each having a different glucose concentration. Thethree calibration chambers C1, C2 and C3 can contain, respectively, thethree calibration fluids. The glucose content of each calibration fluidis different from the other fluids and is a known, i.e. pre-determined,amount.

In some cases, each calibration fluid is made up of water or an aqueoussolution, together with the desired glucose. In some cases, pure wateror an isotonic solution is used, to which the desired glucose content isadded. For example, concentrations of glucose in the calibration fluidscan include zero and concentrations at the upper and lower end of thosewhich are likely to be measured by the sensor. In the example ofcalibration of a glucose sensor for use with intensive care patients,one calibration fluid typically has a zero concentration, whilst theother calibration fluids typically have concentrations of, for example 5mmolL⁻¹ and 10 mmolL¹ respectively. Alternative concentrations could,however, be selected depending on the end use of the sensor.

The order in which the calibration fluids are provided is not critical.In one example, the first calibration fluid has zero glucose content,the second calibration fluid has a glucose concentration of 5 mmoL⁻¹ andan optional third calibration fluid has a concentration of 10 mmoL⁻¹. Inan alternative example, the zero-glucose content calibration fluid isprovided as a preliminary calibration fluid (present within the sensingzone prior to calibration as described further herein) and the first andsecond calibration fluids have non-zero glucose concentrations, e.g. of5 and 10 mmolL⁻¹ respectively.

Each calibration fluid is provided in sterile form Glucose-containingsolutions degrade on sterilisation by heat or gamma-radiation. Thus, thesterile glucose-containing solutions can be prepared by the processdescribed below.

Each calibration chamber can include sufficient calibration fluid tofill sensing zone SZ sufficient for sensing to be carried out. In oneaspect, the calibration fluid itself can be used to flush out anyprevious, used calibration fluid present in the sensing zone. In thiscase, the volume of fluid can be sufficient to fully purge the sensingzone of the used fluid. The volume used can depend on the volume of thesensing zone itself as well as, for example, the inlet. Suitable volumesof calibration fluid are, for example, at least 1 mL, for example atleast 2 mL or at least 5 mL.

The calibration chambers C1, C2 and C3 are, in FIG. 1 , simultaneouslyconnected to an inlet IN to the sensing zone. Alternative embodimentsare also envisaged, however, in which the calibration chambers are notsimultaneously connected to the inlet. For example, the calibrationchambers may be provided in a cartridge, which can be rotated, ortranslated, in order to move each calibration chamber in turn into aposition in which it is connected with the inlet. A one-way valve (notdepicted) may be provided to control fluid exiting the calibrationchamber(s) and to prevent cross-contamination between chambers C1, C2and C3.

Fluid propulsion means, e.g. a pump, can be provided to propelcalibration fluid into the sensing zone. Any suitable means fortransferring calibration fluid may be used. For example, a simpleplunger as depicted at Pu1, Pu2 and Pu3 which pushes calibration fluidout of chamber C1, C2 or C3 respectively into the sensing zone.Alternative means include other types of pump, or a compression zone atthe distal end of the calibration chamber which, when compressed, forcesfluid out of the chamber (e.g. as a pipette).

FIG. 1 depicts one plunger per calibration chamber. However, a singlepropulsion means may be provided, for example in the case where thecalibration fluids are provided in a cartridge as mentioned above. Thepropulsion means may be automated or manual.

After a sensor reading has been taken, calibration fluid can be flushedout of the sensing zone via outlet OUT to waste chamber W. The wastechamber can be separated from the sensing zone by a one-way valve (notdepicted) to prevent cross contamination into the sensing zone.Calibration fluid may be extracted from the sensing zone using separateextraction means, for example by creating a reduced pressure area in thewaste chamber. This can be achieved, for example, by use of a pump, e.g.by extending a plunger provided in waste chamber W to expand the volumeof the chamber. In some cases, separate extraction means are notprovided and instead calibration fluid is flushed out of the sensingzone by the ingress of the next fluid for testing. Thus, for example,once a sensor reading has been taken for the first calibration fluid,the second calibration fluid can be provided to the sensing zone, thisforcing out the first calibration fluid to the waste chamber.

The unit, including the calibration fluids, can be provided in sterileform Thus, the sensor probe and all inner surfaces of the calibrationunit which may come into contact with the calibration fluids can besterile, ensuring that the probe maintains sterility ready for use.Sterility can be provided by use of the preparation process describedfurther herein, which leads to a sealed, sterile calibration unit. Theunit can be adapted such that calibration can be carried out withoutbreaking the seal of the calibration unit and thus maintainingsterility. A seal (e.g. 10) may be opened after calibration to accessthe sensor probe for immediate use.

The calibration unit may include a controller such as a microprocessor,arranged to calibrate the sensor using at least two and typically threesensor output readings. Alternatively, the controller of the sensoritself may be used to carry out the calibration process. In the lattercase, the calibration unit, or disposable sensor probe, may containinformation, for example on precise glucose concentration and volume ofthe calibration fluids, which is readable by the controller (e.g. in barcode form).

As depicted in FIG. 1 , all calibration fluids can be present in thecalibration chambers C1, C2 and C3. However, glucose sensors ofteninclude a hydrogel, for example a hydrogel may be used to immobilise theindicator system. Such sensors are advantageously maintained in hydratedform In an alternative aspect, therefore, one of the calibration fluids(typically a preliminary calibration fluid having zero-glucose content)is provided to the sensing zone at the point of manufacture, for exampleimmediately after sterilisation. This can ensure that a sensing regionof the probe, which is present in the sensing zone, is hydrated duringshipping and storage. This can further reduce the time of calibrationsince the initial sensor output reading can be taken without the need tocarry out any further preliminary steps.

Calibration Process

The calibration can be carried out by providing calibration fluid, e.g.from C1, to the sensing zone via application of pressure, e.g. fromplunger Pu1. The sensor output can then be determined. In the case thatcalibration fluid (typically preliminary calibration fluid which doesnot contain glucose) is provided to the sensing zone prior to storage,this step can be carried out at the point of manufacture. Thecalibration process therefore can be initiated with preliminarycalibration fluid already present within the sensing zone and the firststep in the calibration process can therefore determining sensor outputof the preliminary calibration fluid.

The used calibration fluid can then be flushed from the sensing zone.This may be achieved by creating a reduced pressure in the waste chamberwhich draws calibration fluid out of the sensing zone. In some cases,however, no separate step of removing the used calibration fluid iscarried out. Instead, Pu2 can be depressed forcing the next calibrationfluid to enter the sensing zone, the entry of the next calibration fluidforcing used calibration fluid out of the sensing zone. A second sensoroutput reading can then be taken.

In some cases, these steps can be repeated with a further calibrationfluid, thus providing three sensor output readings which can be used tocalibrate the sensor. In some cases, at least two sensor output readingsare used to calibrate the sensor. In some cases, at least three sensoroutput readings are used to calibrate the sensor. The various steps ofthe calibration process can be automated.

Invasive sensors can, in some cases, operate in a temperature range of35-39 C. However, calibration is normally carried out at roomtemperature. In some cases, sensors calibrated with calibration methods,systems, and devices provided herein can be sensitive to temperaturevariation, in which case a calibration curve generated at roomtemperature may be shifted to a different set of values at, say, 37 C.

The sensor probe described herein may contain a temperature sensor (5 ofFIG. 1 ), such that the temperature at which calibration is carried outcan be determined. If the shift of the calibration curve withtemperature for any particular sensor is known, following generation ofthe calibration curve as described above the curve can be shifted asnecessary to account for the difference in temperature from thatmeasured by the sensor during calibration to 37 C. Alternatively, aheating element (not depicted) may be provided to control thecalibration temperature. For example, the heating element may increasethe temperature within the calibration unit to 37 C prior tocalibration. Alternatively, the temperature may be varied duringcalibration in the manner described in WO2012/164268.

The resulting calibrated sensor may be used to determine the glucosecontent of a sample. In some cases, the user accesses the sensor byremoval of a seal (e.g. 10) or opening the sealed packaging of thecalibration unit. The sensing region of the probe can then inserted intothe sample and the glucose content of the sample determined. The samplemay be an in vitro sample such as a blood or plasma sample, oralternatively the probe is inserted invasively into a human or animalsubject, for example into a blood vessel via a cannular. The sensor maythen be used to determine the blood glucose content.

Preparation of Sterile Calibration Unit

The process for preparing a sterile calibration unit can be carried outaccording to the steps depicted in the flow-chart of FIG. 3 and inschematic steps in FIG. 4 . In some cases, solid glucose 41 and water,or an aqueous solution (e.g. an isotonic solution) intended to form thebasis of the calibration fluid 42 are provided to separate mixingchambers, A and B in a first step. Chambers A and B can be connected insuch a manner that the contents of the chambers are separated duringsterilisation (e.g. to prevent unwanted migration of the contents of thechambers during sterilisation), but can be mixed after sterilisation.For example, chamber A may be connected to, or adapted for connectionto, an inlet to chamber B (or vice versa). In some cases, the chamberscan be separated by a removable seal such that they are in fluidcommunication with one another on removal of the seal, e.g. the chambersmay be separated by one or more valves V1, V2, or removable stoppers,during sterilisation. The chambers being in fluid communication with oneanother means that the water/solution 42 can flow between the twochambers once any stopper/valve means used is opened.

Sterilisation is carried out with the materials in separate mixingchambers A and B. Suitable sterilisation means include heat, e.g.standard autoclaving, or gamma-radiation. Heat, e.g. standardautoclaving is preferred. Following sterilisation, mixing is carriedout, e.g. by opening a valve between the two mixing chambers or removinga stopper between the two chambers. Mixing means may be provided. Forexample, a pump (e.g. a plunger) may be provided to each mixing chamberso that mixing occurs by repeated pumping of solution from one chamberto the other. Alternative mixing means are, however, envisaged, providedthat mixing occurs without loss of sterility. Mixing may be manual orautomated.

All or a portion of the sterile calibration fluid produced can betransferred to a calibration chamber. This may be a separate calibrationchamber which has been pre-sterilised. More conveniently, however,mixing chamber A or B is used as the calibration chamber. FIG. 4 depictsmixing chamber A as the calibration chamber. The chamber is, ifnecessary, sealed (stopper Si) to maintain sterility and to ensurecalibration fluid remains within the chamber. If the chamber is notsealed, attachment to the calibration unit can be carried out understerile conditions.

If only a portion of the calibration fluid is provided in thecalibration chamber, remaining calibration fluid can be left in one ormore mixing chambers, e.g. 44 in mixing chamber B of FIG. 4 .Optionally, the glucose content of the remaining calibration fluid isdetermined. Since the glucose concentration of the remaining fluid isidentical to that of the calibration fluid, this can give a precisefigure for the calibration fluid glucose content, which does not rely oncalculating the concentration from the amount of solid glucose andliquid volume supplied. This has the benefit that inaccuracies in thedispensing of solid glucose or liquid will not affect the calibration.The resulting glucose concentration may be provided to the calibrationunit as a part of the information required for calibration and may beused in the calibration as the glucose concentration of the calibrationfluid.

The above steps can thus provide a glucose-containing calibration fluidin sterile form which can be used in the calibration process describedherein. In some cases, at least two glucose-containing calibrationfluids are required for an accurate calibration of a glucose sensor.Thus, the above steps can be repeated, using a different amount ofglucose 41, and/or a different volume of water/solution 42, to provide afurther calibration fluid having a different glucose concentration. Aglucose-free solution can also be provided. A glucose-free solution maybe sterilised by direct application of heat or gamma-radiation to thecalibration chamber containing the calibration fluid.

The various parts of the calibration unit, including the calibrationchambers, can be placed within a sealed packaging, ready forsterilisation. The sensor probe, e.g. a connector 8 remains outside thepackaging since it does not require sterilisation. Seal 10 may beprovided around the probe to ensure no loss of sterility. The packagingitself can be gas-permeable to allow penetration of the sterilant. Theentire unit can then be sterilised using a surface sterilant such asethylene oxide.

Assembly of the various parts of the calibration unit may be carried outbefore or after sterilisation. In some cases, assembly of the unitoccurs after sterilisation, within the sealed, sterile packaging inorder to maintain sterility. Thus, attachment of the calibrationchambers, e.g. by attachment to inlet IN, may be carried out before orafter sterilisation. Stoppers 51 may be removed from the calibrationchambers prior to attachment. In some cases, the stoppers may be in theform of valves which can be opened at the appropriate time duringcalibration. Similarly, assembly of the sensor probe, e.g. thedisposable probe unit, into the calibration unit by insertion of thesensing region of the probe into the sensing zone, may be carried outbefore or after sterilisation. If before, the calibration unit can begas permeable to allow penetration of the sterilant to the sensor probe.

The calibration unit may be provided alone, or with the sensor probe(e.g. provided as a disposable probe unit) in place in the form of asensor kit. In some cases, the sensor probe is in place in thecalibration unit in order to avoid any loss of sterility by the userhaving to expose a sensor probe and insert it into the calibration unit.Sealing the kit may be conveniently achieved by use of a removable sealwhich the user can peal away to remove the sensor once calibration hasbeen carried out.

The preparation of the unit may optionally include an additional step ofproviding a zero-glucose content calibration fluid to the sensing zone.This can ensure that a sensor probe present in the sensing zone remainshydrated during storage and reduces the time for calibration by the enduser.

1. A process for calibrating a glucose sensor in a sterile environment,the glucose sensor comprising a sensing probe for detecting glucose, theprocess comprising: (i) providing a calibration unit comprising (a) atleast a first and a second sterile calibration fluid, each calibrationfluid having a different concentration of glucose, (b) a sensing zone, asensing region of the sensing probe of the glucose sensor being locatedwithin the sensing zone, wherein the sensing region of the sensing probeis surrounded by a membrane, and (c) a waste chamber for collecting usedcalibration fluid; (ii) providing the first calibration fluid to thesensing zone such that the fluid is in contact with the sensing region,and determining a sensor output; (iii) flushing the first calibrationfluid to the waste chamber; (iv) providing the second calibration fluidto the sensing zone such that the fluid is in contact with the sensingregion, and determining the sensor output; (v) using the sensor outputreadings to calibrate the glucose sensor; wherein the calibrationprocess is carried out under sterile conditions, and wherein saidglucose sensor is an invasive fiber optic glucose sensor containing afluorescent glucose indicator system, the sensor probe being adapted todetect glucose in vivo.
 2. The process according to claim 1, whereinsteps (iii) and (iv) comprise a single step of providing the secondcalibration fluid to the sensing zone, the flow of second calibrationfluid into the sensing zone forcing the first calibration fluid to beflushed into the waste chamber.
 3. The process according to claim 1,wherein a third sterile calibration fluid is provided, having adifferent glucose concentration from both the first and secondcalibration fluids.
 4. The process according to claim 3, wherein theprocess additionally comprises (iiia) flushing the second calibrationfluid to the waste chamber; and (iva) providing the third calibrationfluid to the sensing zone such that the fluid is in contact with thesensing region, and determining the sensor output; and wherein step (v)comprises using all three sensor output readings to calibrate theglucose sensor.
 5. The process according to claim 3, wherein the thirdcalibration fluid is a preliminary calibration fluid having zero glucosecontent and is present in the sensing zone of the calibration unit, andwherein the process comprises (ia) determining the sensor output whilstthe preliminary calibration fluid is in the sensing zone; said step (ia)being carried out prior to step (ii) of providing the first calibrationfluid to the sensing zone.
 6. The process according to claim 1, whichfurther comprises placing the sensing region of the probe in a sampleand determining a glucose content of the sample.
 7. A glucose sensor kitcomprising a sensing probe for detecting glucose and a calibration unit,wherein the calibration unit comprises: a sensing zone having at leastone inlet and at least one outlet, a sensing region of the sensing probebeing located in the sensing zone, wherein the sensing region of thesensing probe is surrounded by a membrane; a first calibration chambercontaining a first sterile calibration fluid, the first calibrationchamber being connected to, or adapted for connection to, an inlet tothe sensing zone; a second calibration chamber containing a secondsterile calibration fluid having a glucose concentration different fromthat of the first calibration fluid, the second calibration chamberbeing connected to, or adapted for connection to, an inlet to thesensing zone; at least one fluid propulsion means for propellingcalibration fluid from the calibration chambers to the sensing zone; awaste chamber configured to collect used calibration fluid which isflushed out of the sensing zone; the glucose sensor kit configured tocarry out calibration of a sensor under sterile conditions, and whereinthe sensing probe is part of an invasive fiber optic glucose sensorcontaining a fluorescent glucose indicator system, and the sensor probeis adapted to detect glucose in vivo.
 8. The glucose sensor kitaccording to claim 7, comprising a controller configured to calibratethe sensor using sensor output readings from the calibration fluids. 9.The glucose sensor kit according to claim 7, wherein the fluidpropulsion means are configured to propel calibration fluid into thesensing zone and simultaneously to flush fluid present in the sensingzone into the waste chamber.
 10. The glucose sensor kit according toclaim 7, further comprising a third calibration chamber containing athird sterile calibration fluid having a glucose concentration differentfrom that of the first and second calibration fluids, the thirdcalibration chamber being connected to, or adapted for connection to, aninlet to the sensing zone.
 11. The glucose sensor kit according to claim7, wherein the sensing zone contains a preliminary sterile calibrationfluid, wherein the preliminary calibration fluid has zero glucosecontent and the first and second calibration fluids have a non-zeroglucose content.
 12. The glucose sensor kit according to claim 7,wherein the first calibration chamber includes a first mixing chamberand a second mixing chamber.
 13. The glucose sensor kit according toclaim 12, wherein a portion of the calibration fluid is provided in thefirst calibration chamber and the remaining fluid is provided withineither the first or the second mixing chamber, and wherein the sensorfurther determines the glucose concentration of the remaining fluid. 14.A calibration unit for calibrating an invasive fiber optic glucosesensor, said sensor containing a fluorescent glucose indicator systemand a sensor probe being adapted to detect glucose in vivo, wherein saidcalibration unit comprises: a sensing zone having at least one inlet andat least one outlet, the sensing zone configured to house a sensingregion of a sensing probe of the glucose sensor, wherein the sensingregion of the sensing probe is surrounded by a membrane; a firstcalibration chamber containing a first sterile calibration fluid, thefirst calibration chamber being connected to, or adapted for connectionto, the inlet to the sensing zone; a second calibration chambercontaining a second sterile calibration fluid having a glucoseconcentration different from that of the first calibration fluid, thesecond calibration chamber being connected to, or adapted for connectionto, the inlet to the sensing zone; at least one fluid propulsion meansfor propelling calibration fluid from the calibration chambers to thesensing zone; a waste chamber configured to collect used calibrationfluid which is flushed out of the sensing zone; the calibration unitbeing configured to carry out calibration of the sensor under sterileconditions.
 15. The calibration unit according to claim 14, furthercomprising a controller configured to calibrate the sensor using sensoroutput readings from the calibration fluids.
 16. The calibration unitaccording to claim 14, further comprising a third calibration chambercontaining a third sterile calibration fluid having a glucoseconcentration different from that of the first and second calibrationfluids, the third calibration chamber being connected to, or adapted forconnection to, the inlet to the sensing zone.
 17. The calibration unitaccording to claim 14, wherein the sensing zone contains a preliminarysterile calibration fluid, wherein the preliminary calibration fluid haszero glucose content and the first and second calibration fluids have anon-zero glucose content.
 18. The calibration unit according to claim14, wherein the first calibration chamber includes a first mixingchamber and a second mixing chamber.
 19. The calibration unit accordingto claim 18, wherein a portion of the calibration fluid is provided inthe first calibration chamber and the remaining fluid is provided withineither the first or the second mixing chamber, and wherein the sensorfurther determines the glucose concentration of the remaining fluid. 20.The calibration unit according to claim 14, wherein the sensing regionof the sensing probe is surrounded by a membrane.